Many genes involved in amino acid metabolism in Bacillus subtilis and related organisms are regulated by the T box mechanism, a novel transcription termination control system. Expression of each gene in this family depends on binding of a specific uncharged tRNA to the nascent RNA transcript, resulting in readthrough of a termination signal that otherwise attenuates downstream gene expression. tRNA recognition requires pairing of the tRNA anticodon with a single codon, the Specifier Sequence, in the leader RNA, as well as pairing of the acceptor end of the tRNA to an antiterminator element that competes with formation of the terminator helix; these pairing are necessary but not sufficient for antitermination. tRNAGly-dependent antitermination of the B. subtilis glyQS gene can be reproduced in a purified system, and binding of tRNAGly to glyQS leader RNA results in structural changes throughout the RNA. The ability of T box RNAs to directly and specifically recognize their cognate effector molecule to regulate gene expression places these RNAs in the riboswitch family of regulatory RNAs. The next project period will be directed toward detailed analysis of the molecular mechanism of tRNA-dependent antitermination, and specific tRNA recognition by T box RNAs. We will focus on RNA elements distinct from the known regions of base-pairing, and functional differences between the glyQS model RNA, which represents a natural deletion variant, and the more common complex T box RNAs that contain additional structural elements, the function of which is unknown. We will also investigate the mechanistic difference between T box elements that operate at the level of transcription termination (found primarily in Firmicutes), and those that operate at the level of translation initiation (found primarily in Actinomycetes, including Mycobacterium sp.). Further characterization of the T box mechanism is essential to the long-term goal of developing novel antimicrobial agents that target this mechanism, which is found in many Gram-positive pathogens. The role of RNA as a regulatory molecule has been increasingly appreciated in recent years, and the T box system represents a unique molecular mechanism for RNA-mediated gene regulation, and a new function for tRNA in the cell. PUBLIC HEALTH RELEVANCE: RNA-mediated regulation has recently emerged as a central player in all organisms. This study is directed toward analysis of the T box system, a unique regulatory mechanism in which RNA transcripts directly sense a specific tRNA molecule to control gene expression. The T box mechanism is widely used in Gram-positive bacteria, including important pathogens, to regulate multiple essential genes, and a long-term goal of this work is the identification of novel antimicrobial agents that inactivate this system, therefore preventing growth.